| dc.contributor.advisor | Tittmann, Kai Prof. Dr. | |
| dc.contributor.author | Schneider, Stefan | |
| dc.date.accessioned | 2014-11-12T10:04:17Z | |
| dc.date.available | 2014-11-12T10:04:17Z | |
| dc.date.issued | 2014-11-12 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0023-9935-6 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-4780 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Functional characterization of transketolase-like proteins and related model systems with respect to thiamin diphosphate mediated chemistry | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Tittmann, Kai Prof. Dr. | |
| dc.date.examination | 2013-12-18 | |
| dc.description.abstracteng | Transketolases (TKTs, E.C 2.2.1.1) are ubiquitous distributed enzymes among all three domains of life. TKTs require thiamin diphosphate (ThDP) and bivalent cations as catalytic cofactors. They fulfill their catalytic function within several metabolic pathways, of which the calvin-cycle of photosynthetic plants and the pentose phosphate pathway (PPP) are the most important ones. The PPP is a cell’s main source of the reductive equivalent nicotine adenine dinucleotid phosphate (NADPH) and ribose 5-phosphate (R5P), the precursor for nucleotide-synthesis. By adjusting the balance of the metabolic flux within the PPP, a cell can adapt towards changing metabolic demands. This metabolic adaption is of particular interest in terms of cancerogenesis, since it has been shown, that cancerous cells seem to modify the generation of NADPH and R5P to force cell dividing capacity. In this regard TKT’s central role in the PPP is of great interest since inhibition of TKT was shown to have growth-inhibitory effects on malignant cells.
Moreover, an isoenzyme of TKT has been described in higher vertebrates, whose proposed function seems to cover the question of an altered PPP-activity. This transketolase-like protein 1 (TKTL1) was shown to be overexpressed on the mRNA- as well as on the protein level in several cancer entities. TKTL1 has been described as having an intrinsic catalytic function, by which it increases the conversion of metabolites within the PPP. Since the hypothesized catalytic function of TKTL1 has not been elucidated in detail, it was the aim of this doctoral thesis to address that question.
It was suggested that TKTL1 follows a reaction mechanism similar to phosphoketolases (XFPKs), another class of ThDP-dependent enzymes of Bifidobacteria. Therefore the reaction mechanism of XFPK of Bifidobacterium breve has been investigated to serve in a comparative manner to enlighten TKTL1’s way of action. XFPK shows a different catalytic activity in comparison to TKTs. Besides sugar phosphates, XFPK also uses inorganic phosphate as substrate. By use of fast kinetic methods and 1 H NMR spectroscopic measurements, it was demonstrated that within the reaction mechanism of XFPK, phosphate not only seems to serve as substrate, but also as accelerator of catalysis. This way of substrate assisted catalysis of phosphate is a unique feature of XFPK among the ThDP-dependent enzyme family.
Furthermore, native TKTL1 was recombinantly expressed and analyzed by spectroscopic methods to enlighten its putative catalytic properties. By usage of established protocols for detection of transketolase activity and mass spectrometry, it could be shown that TKTL1 has no intrinsic transketolase activity. The lack of enzymatic activity is most probably caused by the inability of TKTL1 to bind the ThDP cofactor. This assumption could be underpinned by characterization of a native TKT derived deletion construct. | de |
| dc.contributor.coReferee | Stülke, Jörg Prof. Dr. | |
| dc.contributor.thirdReferee | Dobbelstein, Matthias Prof. Dr. | |
| dc.subject.eng | Transketolase | de |
| dc.subject.eng | TKTL1 | de |
| dc.subject.eng | TKTL2 | de |
| dc.subject.eng | Phosphoketolase | de |
| dc.subject.eng | Thiamin diphosphate | de |
| dc.subject.eng | Substrate assisted catalysis | de |
| dc.subject.eng | Pentose phosphate pathway | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0023-9935-6-5 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 802893228 | |